The annual occurrence of Karenia brevis in the Gulk of Mexico is one of the most significant harmful[unreadable] algal blooms (HABs) in the coastal waters of the United States. This HAB species has a large economic,[unreadable] health, and environmental impact on Gulf coast communities, especially in Florida, through massive fish[unreadable] kills, deaths of marine mammals and birds, human consumption of toxin-contaminated seafood, and[unreadable] respiratory effects from aerosolized toxins. Over the past few decades the incidence of K. brevis blooms[unreadable] appears to be increasing in both frequency and duration. Current government-mandated programs for[unreadable] monitoring this HAB species must rely on microscopic cell counts. These counts are time consuming, have[unreadable] low sample through-put, and require highly-skilled microscopists who are experts at identifying and[unreadable] enumerating Karenia brevis cells of varying morphologies in complex natural phytoplankton assemblages.[unreadable] There is consensus among HAB researchers and public health professionals that monitoring efforts for[unreadable] HABs must evolve towards the use of rapid, accurate, sensitive, and objective molecular-based detection[unreadable] and quantitation methods.[unreadable] The proposed project will first utilize a combination of molecular techniques, such as Fluorescent In Situ[unreadable] Hybridization (FISH) and real-time quantitative polymerase chain reaction (qPCR), with traditional methods[unreadable] (light and fluorescent microscopy) to enumerate Karenia brevis cells in laboratory cultures. The objective of[unreadable] this initial phase is to test the efficacy of, and where feasible optimize, molecular methodologies with a[unreadable] short (3-5 hour) processing time for use in natural populations. Second, we will apply a combination of[unreadable] metabolic measurements (cell division, photosynthesis and respiration rates) and flow cytometry[unreadable] methodologies in cultures, and then natural popualtiosn, to improve our understanding of how[unreadable] environmental factors influence the metabolic state of K. brevis. Third, we will attempt to relate these[unreadable] indices to environmental parameters. The ultimate objective of this research is to facilitate the idenitfication[unreadable] and enumeration of this HAB species, and develop near real-time indices to assess the metabolic state of[unreadable] Karenia brevis in various stages of bloom development.[unreadable] Significance: It is anticipated that the results of this project will enhance our understanding of what[unreadable] factors contribute to Karenia bloom development. It will also synergistically enhance other collaborative[unreadable] HAB research programs through a rapid-response sampling program and sharing of samples and data.